CN103134933A - Test strip used for detecting vibrio parahemolytocus and purpose thereof - Google Patents
Test strip used for detecting vibrio parahemolytocus and purpose thereof Download PDFInfo
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- CN103134933A CN103134933A CN2013100460227A CN201310046022A CN103134933A CN 103134933 A CN103134933 A CN 103134933A CN 2013100460227 A CN2013100460227 A CN 2013100460227A CN 201310046022 A CN201310046022 A CN 201310046022A CN 103134933 A CN103134933 A CN 103134933A
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Abstract
The invention discloses a test strip used for detecting vibrio parahemolytocus. The test strip used for detecting the vibrio parahemolytocus comprises a piece-shaped base material. A first end and a second end are arranged on the piece-shaped base material. A sample cushion, a carbon black cushion, a nitrocellulose film and a water sucking cushion are formed in sequence along the direction from the first end to the second end on the upper surface of the piece-shaped base material. A detection line is formed on the nitrocellulose film and is close to one side of the first end of the piece-shaped base material. A quality control line is formed on the nitrocellulose film and is close to one side of the second end of the piece-shaped base material. The carbon black cushion is formed by carbon black particles. At lest one part of the carbon black particles comprise anti- vibrio parahemolytocus monoclonal immune body. The detection line is formed by anti- vibrio parahemolytocus polyclone immune body on the nitrocellulose film. The quality control line is formed by goat anti mouse immune body attached on the nitrocellulose film. According to the test strip used for detecting the vibrio parahemolytocus, the vibrio parahemolytocus can be effectively detected.
Description
Technical field
The present invention relates to biological technical field, concrete, the present invention relates to test strips for detection of vibrio parahemolyticus and uses thereof, especially relate to the fast detecting of vibrio parahemolyticus in marine product.
Background technology
Vibrio parahemolyticus (Vibrio Parahemolyticus, VP) be a kind of common food-borne pathogens, mainly be present in the marine products such as inkfish, ocean fish, extra large shrimp, sea crab, jellyfish, its food poisoning that causes is clinically take stomachache, vomiting, diarrhoea etc. as cardinal symptom.This disease is many betides the coastland in summer and autumn, causes collective's morbidity.In recent years due to the seafood air transport, the inland city case is also cumulative many, and particularly in the coastland, the number several and that relate to of having fallen ill is even more serious than other areas of China, must set up reliable method for quick, monitor for a long time the pollution situation of vibrio parahemolyticus in aquatic products.Traditional microorganism culture identification method is laboratory detection method commonly used, but length consuming time, the shortcomings such as complex operation can not satisfy the requirement of fast detecting vibrio parahemolyticus.Although the technology such as PCR, DNA probe has been widely used in the detection of vibrio parahemolyticus, matrix effect disturbs larger to testing result.In recent years, the enzyme-linked immune analytic method take immunological response as the basis is rapid in development aspect microorganism detection, and especially immune chromatography method has more embodied the rapidity of method.Yet the means for detection of vibrio parahemolyticus still remain to be improved at present.
Summary of the invention
The present invention one of is intended to solve the problems of the technologies described above at least to a certain extent or provides at least a kind of useful business to select.For this reason, one object of the present invention is to propose a kind of test strips that can be quick, easy, sensitive, special, cheap.
In one aspect of the invention, the present invention proposes a kind of test strips for detection of vibrio parahemolyticus.Comprise: flat substrates, described flat substrates have first end and the second end, and at the upper surface of described flat substrates, are formed with successively sample pad, carbon black pad, nitrocellulose filter and adsorptive pads along the direction of first end to the second end; Detection line, described detection line are formed on described nitrocellulose filter the side near described flat substrates first end; Nature controlling line, described nature controlling line is formed on described nitrocellulose filter the side near described flat substrates the second end, wherein, described carbon black pad is formed by carbon black pellet, and at least a portion of wherein said carbon black pellet is coated with anti-vibrio parahemolyticus monoclonal antibody; Described detection line is formed by the anti-vibrio parahemolyticus polyclonal antibody that is attached on described nitrocellulose filter; And described nature controlling line is formed by the sheep anti-mouse antibody that is attached on described nitrocellulose filter.The inventor finds, can significantly improve the sensitivity carbon black test strips of test strips by adopting carbon black pellet, easy to use, be easy to carry, visual result, preservation be simple, testing result only needs 10 minutes, is limited to 1 * 10 under detection
3Cfu/mL.This detects vibrio parahemolyticus for basic unit convenience is provided, the traditional technique in measuring of comparing, and this test strips has been saved a large amount of manpower and financial resources, has great promotion and use value.In research process, inventor's discovery, when adopting carbon black granules as anti-vibrio parahemolyticus labeling of monoclonal antibody thing, the sensitivity of test strips reaches 1 * 10
3Cfu/mL, and when adopting traditional colloid gold particle as the label of anti-vibrio parahemolyticus monoclonal antibody, the sensitivity of test strips only has 1 * 10
4Cfu/mL.By contrast, adopt carbon black pellet than adopting collaurum and serve as a mark thing, the sensitivity of test strips exceeds an order of magnitude.In addition, can also have following beneficial effect one of at least for detection of the test strips of vibrio parahemolyticus according to an embodiment of the invention: (1) is highly sensitive, and a small amount of bacterium just can detect; (2) high specificity; (3) good stability; (4) simple to operate, need not instrument and equipment; (5) being easy to transportation stores.
According to embodiments of the invention, be used to form the material of flat substrates and be not particularly limited, preferred, described flat substrates is formed by PVC.Thus, can further improve detection efficiency for detection of the test strips of vibrio parahemolyticus.
According to embodiments of the invention, described carbon black pellet prepares through the following steps: after combustion synthesis in air, residue is collected in glass flask with toluene, adds toluene and heating to boil and filters after being cooled to normal temperature, obtains filter residue; And described filter residue was heat-treated under 260 degrees centigrade 12 hours, in order to obtain described carbon black pellet.Thus, can further improve detection efficiency for detection of the test strips of vibrio parahemolyticus.
According to embodiments of the invention, described filter residue was heat-treated under 260 degrees centigrade 12 hours.Thus, can further improve detection efficiency for detection of the test strips of vibrio parahemolyticus.
According to embodiments of the invention, before described filter residue is heat-treated, utilize organic solvent that described filter residue is washed and vacuum drying, wherein, described organic solvent is be selected from toluene, dimethyl formamide and hexane at least a.Thus, can further improve detection efficiency for detection of the test strips of vibrio parahemolyticus.
According to embodiments of the invention, described anti-vibrio parahemolyticus monoclonal antibody is to be coated in through the following steps on described carbon black pellet: described carbon black pellet is mixed with described anti-parahemolyticas monoclonal antibody, in order to obtain the first potpourri; And add glutaraldehyde in described the first potpourri, in order to make described anti-parahemolyticas monoclonal antibody and described carbon black pellet be coupled.Thus, can further improve detection efficiency for detection of the test strips of vibrio parahemolyticus.
According to embodiments of the invention, described anti-vibrio parahemolyticus monoclonal antibody is to be coated in through the following steps on described carbon black pellet: the 5.0mg carbon black is added in the PBS of 50mL0.01M pH7.2, slowly add the 250 anti-parahemolyticas monoclonal antibodies of μ g to react, stirring reaction 24h under normal temperature; The glutaraldehyde that adds 50mL25 % by weight pH7.2, stirring reaction 120min, 6000 * g, centrifugal 10min; Abandon supernatant, precipitation is resuspended with the PBS of 0.01M pH7.2, crosses post through Sepharose 6B-CL, removes carbon black and the glutaraldehyde of not coupling, in order to obtain the carbon black pellet of the anti-vibrio parahemolyticus monoclonal antibody of pan coating.Thus, can further improve detection efficiency for detection of the test strips of vibrio parahemolyticus.
In a second aspect of the present invention, the invention allows for the foregoing purposes of test strips in detecting vibrio parahemolyticus for detection of vibrio parahemolyticus.
Additional aspect of the present invention and advantage part in the following description provide, and part will become obviously from the following description, or recognize by practice of the present invention.
Description of drawings
Above-mentioned and/or additional aspect of the present invention and advantage are from obviously and easily understanding becoming the description of embodiment in conjunction with following accompanying drawing, wherein:
Fig. 1 is according to an embodiment of the invention for detection of the planar structure schematic diagram of the test strips of vibrio parahemolyticus;
Fig. 2 is according to an embodiment of the invention for detection of the side structure schematic diagram of the test strips of vibrio parahemolyticus;
Fig. 3 is according to an embodiment of the invention for detection of the negative findings figure of the test strips of vibrio parahemolyticus;
Fig. 4 is according to an embodiment of the invention for detection of the positive findings figure of the test strips of vibrio parahemolyticus;
Fig. 5 is according to an embodiment of the invention for detection of the null result figure of the test strips of vibrio parahemolyticus.
Embodiment
The below describes embodiments of the invention in detail, and the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or the element with identical or similar functions from start to finish.Be exemplary below by the embodiment that is described with reference to the drawings, be intended to for explanation the present invention, and can not be interpreted as limitation of the present invention.
in description of the invention, it will be appreciated that, term " " center ", " vertically ", " laterally ", " length ", " width ", " thickness ", " on ", D score, " front ", " afterwards ", " left side ", " right side ", " vertically ", " level ", " top ", " end ", " interior ", " outward ", " clockwise ", orientation or the position relationship of indications such as " counterclockwise " are based on orientation shown in the drawings or position relationship, only the present invention for convenience of description and simplified characterization, rather than device or the element of indication or hint indication must have specific orientation, with specific orientation structure and operation, therefore can not be interpreted as limitation of the present invention.
In addition, term " first ", " second " only are used for describing purpose, and can not be interpreted as indication or hint relative importance or the implicit quantity that indicates indicated technical characterictic.Thus, one or more these features can be expressed or impliedly be comprised to the feature that is limited with " first ", " second ".In description of the invention, the implication of " a plurality of " is two or more, unless clear and definite concrete restriction is separately arranged.
In the present invention, unless clear and definite regulation and restriction are separately arranged, broad understanding should be done in the terms such as term " installation ", " being connected ", " connection ", " fixing ", for example, can be to be fixedly connected with, and can be also to removably connect, or connect integratedly; Can be mechanical connection, can be also to be electrically connected to; Can be directly to be connected, also can indirectly be connected by intermediary, can be the connection of two element internals.For the ordinary skill in the art, can understand as the case may be above-mentioned term concrete meaning in the present invention.
In the present invention, unless clear and definite regulation and restriction are separately arranged, First Characteristic Second Characteristic it " on " or D score can comprise that the first and second features directly contact, can comprise that also the first and second features are not directly contacts but by the other feature contact between them.And, First Characteristic Second Characteristic " on ", " top " and " above " comprise First Characteristic directly over Second Characteristic and oblique upper, or only represent that the First Characteristic level height is higher than Second Characteristic.First Characteristic Second Characteristic " under ", " below " and " below " comprise First Characteristic under Second Characteristic and tiltedly, or only represent that the First Characteristic level height is less than Second Characteristic.
Below, at first with reference to figure 1 and Fig. 2, the test strips for detection of vibrio parahemolyticus is described.With reference to figure 1 and Fig. 2, should comprise flat substrates 7 for detection of the test strips of vibrio parahemolyticus, this flat substrates 7 has first end and the second end, and at the upper surface of this flat substrates 7, be formed with successively sample pad 1, carbon black pad 2, nitrocellulose filter 3 and adsorptive pads 6 along the direction of first end to the second end.
According to embodiments of the invention, also be formed with detection line 4 and nature controlling line 5 on nitrocellulose filter 3.Wherein, detection line 4 is formed on nitrocellulose filter 3 side near flat substrates 7 first ends.Nature controlling line 5 is formed on nitrocellulose filter 3 side near flat substrates 7 second ends.
According to embodiments of the invention, carbon black pad 2 is formed by carbon black pellet, and wherein at least a portion of carbon black pellet is coated with anti-vibrio parahemolyticus monoclonal antibody.Detection line 4 is formed by the anti-vibrio parahemolyticus polyclonal antibody that is attached on nitrocellulose filter 3.Nature controlling line 5 is formed by the sheep anti-mouse antibody that is attached on nitrocellulose filter 3.
According to embodiments of the invention, described anti-vibrio parahemolyticus monoclonal antibody is as the carbon black labelled antibody, anti-vibrio parahemolyticus polyclonal antibody is to be sprayed on detection line 4 places, and two antibody can only form sandwich complex as intermediate by vibrio parahemolyticus, i.e. double antibodies sandwich.
According to embodiments of the invention, the detection principle of test strips is add liquid to be checked in the ELISA test strip hole after, if contain vibrio parahemolyticus in liquid to be checked, the polyclonal antibody at the monoclonal anti physical efficiency of carbon black mark and detection line 4 places forms double antibodies sandwich so, increase along with catching quantity, namely at the aobvious black in detection line 4 places, remaining thalline-when the monoclonal anti nanocrystal composition continues to migrate to nature controlling line 5 place, compound can be caught by the sheep anti-mouse antibody specificity at nature controlling line 5 places, increase along with catching quantity, i.e. the aobvious black of nature controlling line.If do not contain vibrio parahemolyticus in liquid to be checked, the monoclonal antibody of carbon black mark can not combine with the polyclonal antibody at detection line 4 places so, namely at the not aobvious black in detection line 4 places, when thalline-when the monoclonal anti nanocrystal composition continues to migrate to nature controlling line 5 place, this antibody capable is caught by the sheep anti-mouse antibody specificity at nature controlling line 5 places, increase along with catching quantity, i.e. the aobvious black in nature controlling line 5 places.
According to embodiments of the invention, be used to form the material of flat substrates and be not particularly limited, preferred, described flat substrates is formed by PVC.Thus, can further improve detection efficiency for detection of the test strips of vibrio parahemolyticus.
According to embodiments of the invention, described carbon black pellet prepares through the following steps: after combustion synthesis in air, residue is collected in glass flask with toluene, adds toluene and heating to boil and filters after being cooled to normal temperature, obtains filter residue; And described filter residue was heat-treated under 260 degrees centigrade 12 hours, in order to obtain described carbon black pellet.Thus, can further improve detection efficiency for detection of the test strips of vibrio parahemolyticus.
According to embodiments of the invention, described filter residue was heat-treated under 260 degrees centigrade 12 hours.Thus, can further improve detection efficiency for detection of the test strips of vibrio parahemolyticus.
According to embodiments of the invention, before described filter residue is heat-treated, utilize organic solvent that described filter residue is washed and vacuum drying, wherein, described organic solvent is be selected from toluene, dimethyl formamide and hexane at least a.Thus, can further improve detection efficiency for detection of the test strips of vibrio parahemolyticus.
According to embodiments of the invention, described anti-vibrio parahemolyticus monoclonal antibody is to be coated in through the following steps on described carbon black pellet: described carbon black pellet is mixed with described anti-parahemolyticas monoclonal antibody, in order to obtain the first potpourri; And add glutaraldehyde in described the first potpourri, in order to make described anti-parahemolyticas monoclonal antibody and described carbon black pellet be coupled.Thus, can further improve detection efficiency for detection of the test strips of vibrio parahemolyticus.
According to embodiments of the invention, described anti-vibrio parahemolyticus monoclonal antibody is to be coated in through the following steps on described carbon black pellet: the 5.0mg carbon black is added in the PBS of 50mL0.01M pH7.2, slowly add the 250 anti-parahemolyticas monoclonal antibodies of μ g to react, stirring reaction 24h under normal temperature; The glutaraldehyde that adds 50mL25 % by weight pH7.2, stirring reaction 120min, 6000 * g, centrifugal 10min; Abandon supernatant, precipitation is resuspended with the PBS of 0.01M pH7.2, crosses post through Sepharose 6B-CL, removes carbon black and the glutaraldehyde of not coupling, in order to obtain the carbon black pellet of the anti-vibrio parahemolyticus monoclonal antibody of pan coating.Thus, can further improve detection efficiency for detection of the test strips of vibrio parahemolyticus.
According to embodiments of the invention, the main hemotoxin TLH of described anti-parahemolyticas monoclonal antibody specificity identification vibrio parahemolyticus.Thus, can further improve detection efficiency for detection of the test strips of vibrio parahemolyticus.
According to embodiments of the invention, the main hemotoxin TDH of described anti-vibrio parahemolyticus polyclonal antibody identification vibrio parahemolyticus.Thus, can further improve detection efficiency for detection of the test strips of vibrio parahemolyticus.
In a second aspect of the present invention, the invention allows for the foregoing purposes of test strips in detecting vibrio parahemolyticus for detection of vibrio parahemolyticus.
Below by specific embodiment, the test strips for detection of vibrio parahemolyticus of the present invention is described.The following examples are only illustrative, do not limit the present invention in any way.
(1) preparation of vibrio parahemolyticus monoclonal antibody
By clone recombination to construct main hemotoxin TLH and the prokaryotic expression carrier pET30a/tlh of vibrio parahemolyticus,
Change it in E.coli Bl21 abduction delivering recombinant protein His-TLH, adopt affinity chromatography method purifying.The deactivation thalline, the preparation immunogene, the amount of every BALB/c mouse injection TLH is 100 μ g, immunity four times, every two weeks of minor tick, adopting the ELISA method to measure mouse tires, after reaching requirement when tiring, the myeloma cell who gets mouse boosting cell and recovery in advance mix (10:1 ~ 5:1), centrifugal after, dropwise the PEG of 37 ℃ of preheatings merges, the incomplete nutrient culture media that adds 37 ℃ of preheatings after static 90s, centrifugal rear resuspended with the HAT nutrient culture media, join in the 96 porocyte culture plates that contain in advance feeder cells, the 5%CO2 incubator is cultivated.After 3 days, observation of cell merges situation, changes half nutrient solution after 5 ~ 6 days, and 7 ~ 10 days sucking-off cell conditioned medium liquid carries out ELISA and detects, through repeatedly detecting and cloning, filter out can stably excreting vibrio parahemolyticus monoclonal antibody cell line.Cultivate by expansion, adopt whiteruss that mouse peritoneal is injected, the hybridoma of 7 ~ 10 days pneumoretroperitoneum injection serum free mediums dilution, observe mouse web portion after a couple of days, when mouse web portion obviously protuberance and mouse state when relatively poor, draw ascites with asepsis injector from mouse peritoneal, ascites is adopted the monoclonal antibody that obtains vibrio parahemolyticus after sad-ammonium sulfate purifying.
(2) preparation of parahemolyticas polyclonal antibody
By clone's recombination to construct main hemotoxin TDH and the prokaryotic expression carrier pET30a/tdh of vibrio parahemolyticus, change it in E.coli Bl21 abduction delivering recombinant protein His-TDH, adopt affinity chromatography method purifying.The standby immunogene of inactivated bacteria system, every New Zealand white rabbit immunizing dose is 1mg, immunity four times, every two weeks of minor tick, during adopt ELISA to measure antiserum titre, a week after last immunity, the femoral artery blood sampling is taken out after serum is separated out in the blood aggegation, and-20 ℃ save backup.Adopt caprylic acid-ammonium to carry out purifying to serum, namely get anti-vibrio parahemolyticus polyclonal antibody.
(3) preparation of carbon black pellet
After 500mL toluene was aloft burnt, carbon black was collected in glass flask, added the heating of 50mL toluene to boil 20min, was cooled to normal temperature, filtered.Add 50mL toluene repeated washing residue 5 times, then respectively wash 5 times with dimethyl formamide and hexane respectively, filter vacuum drying.Then with the high temperature action 12h of reaction product at 260 ℃, namely obtain the Powdered carbon black of mute black.
(4) carbon black labeled monoclonal antibody
The 5.0mg carbon black is added in 50mL0.01M PBS (pH7.2), slowly add the 250 anti-parahemolyticas monoclonal antibodies of μ g to react, stirring reaction 24h under normal temperature.The glutaraldehyde (pH7.2) that adds 50mL25%, stirring reaction 120min, 6000 * g, centrifugal 10min.Abandon supernatant, precipitation is resuspended with 0.01M PBS (pH7.2), crosses post through Sepharose6B-CL, removes carbon black and the glutaraldehyde of not coupling, saves backup with the phosphate buffer of 1%BSA, 25% glycerine and 0.05% Sodium azide.
(5) preparation of carbon black pad
(after pH7.4PBS+1%Tween20+0.1%PVA+7% sucrose+1%BSA) carries out pre-service to blank pad (glass fibre), the carbon black label is sprayed on the pad of 40cm * 6cm 37 ℃ of dry 2h with pretreatment fluid.
(6) processing of detection line and nature controlling line on nitrocellulose filter
Spray anti-vibrio parahemolyticus polyclonal antibody as detection line at 0.8 ~ 0.9cm place, distance nitrocellulose filter CN140 lower end, 0.9 ~ 1.0cm place sprays sheep anti-mouse antibody as nature controlling line apart from the nitrocellulose filter upper end, 37 ℃ of dry 2h.
(7) assembling of carbon black test strips
Test strips is comprised of sample pad, carbon black pad, nitrocellulose filter, adsorptive pads and PVC backing.Altogether be divided into 4 parts: sample application zone, label land, colour developing district, suction zones.The left end of test strips is sample application zone, and the loading volume is 100 μ L.The mark land is the carbon black labeled monoclonal antibody, colour developing district for nitrocellulose filter coated upper vibrio parahemolyticus polyclonal antibody successively and sheep anti-mouse antibody respectively as detection line and nature controlling line.Suction zones is that thieving paper sticks on the part nitrocellulose filter.This test strips is utilized immunochromatography technique and double antibodies sandwich method principle, comes the negative and positive of judgement sample by the colour developing of observing on detection line and nature controlling line.When detection line does not develop the color, the nature controlling line colour developing judges that sample is negative; When the detection line colour developing, the nature controlling line colour developing judges that sample is positive; When nature controlling line does not develop the color, judge that test strips lost efficacy.Whole test strips is contained in during the north material of PVC gets stuck, and test strips is loaded in aluminium foil bag and seals, and add the silica gel particle drying agent, but in room temperature, long preservation is standby.
(8) the specific mensuration of test strips
Select vibrio parahemolyticus and other 5 kinds of common pathogenic bacteria: Escherichia coli O 157: H7, salmonella, staphylococcus aureus, Listeria monocytogenes, Shigella all are diluted to 1 * 10
6The cfu/mL bacteria suspension is got respectively the sample application zone that 100 μ L are added drop-wise to test strips, observations in 10min.Detection line colour developing on the quick measuring card of measuring vibrio parahemolyticus is only arranged, the nature controlling line colour developing, namely positive, on other 5 kinds of common pathogen quick measuring cards, detection line is colourless, and only nature controlling line colour developing illustrates that this test strips has very strong specificity to vibrio parahemolyticus.
(9) sensitivity determination of test strips
With vibrio parahemolyticus from 1 * 10
7Cfu/mL is diluted to 1 * 10
1Cfu/mL gets respectively 100 μ L in well, result of determination after 10min.According to the colour developing result of test strips, can judge that test strips can detect 1 * 10
3The bacterium liquid of cfu/mL is when bacterial concentration is 1 * 10
2During cfu/mL, the colour developing of the detectability of test strips is lower, and therefore, the sensitivity of this test strips is 1 * 10
3The vibrio parahemolyticus of cfu/mL.
(1) sample preparation
Doubtful inkfish is smashed to pieces put into centrifuge tube, add sterilized water to soak 30min, then shake 3min, draw supernatant.
(2) increase in advance bacterium
Supernatant is added in the vibrio parahemolyticus selective medium, and nutrient culture media is mixed protein peptone 2% (tryptone 1%, fish peptone 1%), yeast extract 0.2%, NaCl 3.5%, pH 8.6; 37 ℃ of shaking tables (100r/min) are cultivated 8h, double-deck kraft sealing.
(3) sample detection
Tear aluminium foil bag before detection, take out test strips, lie against on operator's console, placed 3 minutes, supernatant to be measured is directly added well, observe testing result after 10min.
(4) result is judged
With reference to figure 3 ~ 5, if detection line does not develop the color, nature controlling line colour developing shows not contain vibrio parahemolyticus in liquid to be measured (in sample, the concentration of vibrio parahemolyticus is lower than 1 * 10
3Cfu/mL), namely negative; If the detection line colour developing, the nature controlling line colour developing shows in liquid to be measured to contain vibrio parahemolyticus that namely positive (in sample, the concentration of vibrio parahemolyticus is higher than 1 * 10
3Cfu/mL); If detection line is indistinct, the nature controlling line colour developing illustrates that containing vibrio parahemolyticus in liquid to be measured (contain vibrio parahemolyticus in sample, but concentration is lower than 1 * 10 lower than detecting limit value
3Cfu/mL); If nature controlling line does not develop the color, illustrate that test strips is invalid, testing result can not be as judgment basis.
(5) colloidal gold strip detects vibrio parahemolyticus
Make colloidal gold strip according to the principle of the invention, carry out the vibrio parahemolyticus detection by example 2 steps, with this sensitivity of judging colloidal gold strip, the results are shown in Table 1.According to measurement result, the sensitivity of this colloidal gold strip is 1 * 10
4Cfu/mL.
Table 1 colloidal gold strip is measured vibrio parahemolyticus
In the description of this instructions, the description of reference term " embodiment ", " some embodiment ", " example ", " concrete example " or " some examples " etc. means to be contained at least one embodiment of the present invention or example in conjunction with specific features, structure, material or the characteristics of this embodiment or example description.In this manual, the schematic statement of above-mentioned term not necessarily referred to identical embodiment or example.And the specific features of description, structure, material or characteristics can be with suitable mode combinations in any one or more embodiment or example.
Although the above has illustrated and has described embodiments of the invention, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art is not in the situation that break away from principle of the present invention and aim can change above-described embodiment within the scope of the invention, modification, replacement and modification.
Claims (10)
1. the test strips for detection of vibrio parahemolyticus, is characterized in that, comprising:
Flat substrates, described flat substrates have first end and the second end, and at the upper surface of described flat substrates, are formed with successively sample pad, carbon black pad, nitrocellulose filter and adsorptive pads along the direction of first end to the second end;
Detection line, described detection line are formed on described nitrocellulose filter the side near described flat substrates first end;
Nature controlling line, described nature controlling line are formed on described nitrocellulose filter the side near described flat substrates the second end,
Wherein,
Described carbon black pad is formed by carbon black pellet, and at least a portion of wherein said carbon black pellet is coated with anti-vibrio parahemolyticus monoclonal antibody;
Described detection line is formed by the anti-vibrio parahemolyticus polyclonal antibody that is attached on described nitrocellulose filter; And
Described nature controlling line is formed by the sheep anti-mouse antibody that is attached on described nitrocellulose filter.
2. the test strips for detection of vibrio parahemolyticus according to claim 1, is characterized in that, described flat substrates is formed by PVC.
3. the test strips for detection of vibrio parahemolyticus according to claim 1, is characterized in that, described carbon black pellet prepares through the following steps:
After combustion synthesis in air, residue is collected in glass flask with toluene, adds toluene and heating to boil and filters after being cooled to normal temperature, obtains filter residue; And
Described filter residue was heat-treated under 260 degrees centigrade 12 hours, in order to obtain described carbon black pellet.
4. the test strips for detection of vibrio parahemolyticus according to claim 1, is characterized in that, described filter residue was heat-treated under 260 degrees centigrade 12 hours.
5. the test strips for detection of vibrio parahemolyticus according to claim 1, is characterized in that, before described filter residue is heat-treated, utilizes organic solvent that described filter residue is washed and vacuum drying,
Wherein, described organic solvent is be selected from toluene, dimethyl formamide and hexane at least a.
6. the test strips for detection of vibrio parahemolyticus according to claim 1, is characterized in that, described anti-vibrio parahemolyticus monoclonal antibody is to be coated in through the following steps on described carbon black pellet:
Described carbon black pellet is mixed with described anti-parahemolyticas monoclonal antibody, in order to obtain the first potpourri; And
Add glutaraldehyde in described the first potpourri, in order to make described anti-parahemolyticas monoclonal antibody and described carbon black pellet be coupled.
7. the test strips for detection of vibrio parahemolyticus according to claim 1, is characterized in that, described anti-vibrio parahemolyticus monoclonal antibody is to be coated in through the following steps on described carbon black pellet:
The 5.0mg carbon black is added in the PBS of 50mL0.01M pH7.2, slowly add the 250 anti-parahemolyticas monoclonal antibodies of μ g to react, stirring reaction 24h under normal temperature;
The glutaraldehyde that adds 50mL25 % by weight pH7.2, stirring reaction 120min, 6000 * g, centrifugal 10min;
Abandon supernatant, precipitation is resuspended with the PBS of 0.01M pH7.2, crosses post through Sepharose 6B-CL, removes carbon black and the glutaraldehyde of not coupling, in order to obtain the carbon black pellet of the anti-vibrio parahemolyticus monoclonal antibody of pan coating.
8. the test strips for detection of vibrio parahemolyticus according to claim 1, is characterized in that, the main hemotoxin TLH of described anti-parahemolyticas monoclonal antibody specificity identification vibrio parahemolyticus.
9. the test strips for detection of vibrio parahemolyticus according to claim 1, is characterized in that, the main hemotoxin TDH of described anti-vibrio parahemolyticus polyclonal antibody identification vibrio parahemolyticus.
10. the purposes of the described test strips for detection of vibrio parahemolyticus of claim 1 ~ 9 any one in detecting vibrio parahemolyticus.
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Denomination of invention: Test strip used for detecting vibrio parahemolytocus and purpose thereof Effective date of registration: 20180730 Granted publication date: 20150701 Pledgee: Bank of Beijing, Limited by Share Ltd, Nanchang branch Pledgor: Jiangxi Sino German bioengineering Limited by Share Ltd Registration number: 2018990000621 |
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Date of cancellation: 20190927 Granted publication date: 20150701 Pledgee: Bank of Beijing, Limited by Share Ltd, Nanchang branch Pledgor: Jiangxi Sino German bioengineering Limited by Share Ltd Registration number: 2018990000621 |